In recent years a handful of Aequorea green FP (GFP) homologues cloned from Anthozoan organisms have been reported to undergo irreversible photoconversion from a green fluorescent to a red fluorescent species upon illumination with light of approximately 400 nm. To date, the naturally occurring photoconvertible proteins that have received the most attention are Kaede from coral Trachyphyllia geoffroyi (Ando et al., 2002), EosFP from stony coral Lobophyllia hemprichii (Wiedenmann et al., 2004), and Dendra from octocoral Dendronephthya sp. (Gurskaya et al., 2006). It has also been demonstrated that a non-photoconvertible FP can be engineered to be photoconvertible FPs. Specifically, the photoconvertible FP known as KikGR was engineered from the green fluorescent KikG of the coral Favia favus (Tsutsui et al., 2005).
All green-to-red photoconvertible FPs characterized to date share a common His-Tyr-Gly-derived chromophore structure, and a common photoconversion mechanism (FIG. 1) (Mizuno et al., 2003). The newly synthesized protein first folds into the characteristic beta-barrel structure that defines the Aequorea GFP superfamily (Ormo et al., 1996), and undergoes the steps of post-translational modification that lead to the formation of a green fluorescent chromophore with a conjugated system identical to that of the Aequorea GFP chromophore. The chromophore can exist in either its neutral phenol form or the anionic phenolate form (FIG. 1). Exactly where the equilibrium between these two forms lies is dependent on the local microenvironment of the chromophore (as determined by the amino acid substitutions in close proximity to it) and the pH of the solution. The green-to-red photoconvertible FPs are distinguished from their non-photoconvertible brethren, by the respective consequences of exciting the neutral form which absorbs most strongly at ˜400 nm. In wild-type Aequorea GFP the excited state of the neutral form undergoes excited state proton transfer to form the anionic form, which then emits green fluorescence (Chattoraj et al., 1996). An Aequorea GFP variant that does fluoresce from the neutral form of the excited state has been engineered (Tsutsui et al., 2009). In the case of the green-to-red photoconvertible FPs, excitation of the neutral form leads to a break of the polypeptide chain through the effective beta-elimination of the residue that immediately precedes the chromophore-forming His-Tyr-Gly tripeptide (Mizuno et al., 2003; Tsutsui et al., 2009). This elimination reaction results in the installation of a new double bond between C-alpha and C-beta of the His residue, placing the side chain imidazole into conjugation with the remainder of the avGFP-type chromophore. This extended conjugation decreases the HOMO-LUMO gap and thus shifts the emission into the orange-to-red region of the visible spectrum. Photoconversion via alternate mechanisms has been observed in other color classes of FP (Chudakov et al., 2004; Kremers et al., 2009).
Kaede, the first example of a FP that can undergo an irreversible green-to-red photoconversion upon illumination with UV light, was initially described by Miyawaki and coworkers in 2002 (Ando et al., 2002). Unfortunately, the range of potential applications for Kaede remains limited by the fact that it is an obligate tetramer (Hayashi et al., 2007), and no monomeric variants have been reported. Unlike monomeric FPs, tetrameric FPs are generally detrimental to the proper trafficking and localization of recombinant fusion proteins (Campbell et al., 2002). To produce a monomeric green-to-red photoconvertible FP, the same workers appear to have had more success with engineered variants of the tetrameric KikGR FP which is substantially brighter and more efficiently photoconverted than Kaede (Tsutsui et al., 2005). A monomeric version of KikGR, known as mKikGR, has recently been reported (Habuchi et al., 2008).
The two other green-to-red photoconvertible FPs, EosFP and Dendra (Gurskaya et al., 2006; Wiedenmann et al., 2004), have both been subjected to protein engineering to convert the wild-type tetramers into monomers (Adam et al., 2009; McKinney et al., 2009). However, it is apparent that in both eases the ‘monomeric’ FP does retain some tendency to form dimers at high concentrations (McKinney et al., 2009). The monomeric variant of EosFP, known as mEos, was created through the introduction of 2 point mutations that disrupted the protein-protein interfaces of the tetrameric species (Nienhaus et al., 2005; Wiedenmann et al., 2004). Expression of mEos temperatures of less than 30 degrees C. is problematic (Wiedenmann et al., 2004), but an effectively monomeric tandem dimer variant does express well at 37 degrees C. (Nienhaus et al., 2006). The problem of poor expression of mEos at 37 degrees C. has been overcome with the engineering of mEos2 (McKinney et al., 2009) through the targeted substitution of residues with solvent exposed side chains. Although mEos2 has been reported to retain some propensity for dimer formation, this property does not appear to have adverse effects on the subcellular targeting of a variety of fusion proteins (McKinney et al., 2009).
As with mEos, Dendra is a monomeric green-to-red photoconvertible FP derived from the wild-type tetrameric DendGFP (Gurskaya et al., 2006). Further optimization of Dendra produced the Dendra2 variant, which is claimed to be brighter and faster maturing (Chudakov et al., 2004). Unlike other members of the class of green-to-red photoconvertible FPs that require UV illumination to promote photoconversion, Dendra can be converted to its red fluorescent state by either cyan or UV wavelengths of light. Although Dendra2 generally behaves as a monomer in relatively dilute solutions, it was observed to reconstitute a protein-protein interface very similar to the typical AC dimer interface observed in crystal structures of tetrameric FPs (Adam et al., 2009). It has been reported that Dendra2 can fluoresce from either the neutral or anionic forms of the photoconverted species.